Development of a highly specific LAMP assay for detection of Sarcocystis tenella and Sarcocystis gigantea in sheep

IF 1.8 3区 医学 Q2 PARASITOLOGY Parasitology Research Pub Date : 2024-09-14 DOI:10.1007/s00436-024-08349-0
Yajie Chen, Jing Peng, Zifu Zhu, Wen Zhang, Lifang Wang, Jianhai Xu, Qun Liu, Jing Liu
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Abstract

Sarcocystis infection in sheep has caused significant economic losses in the livestock industry, and the genetic similarity among Sarcocystis species highlights the need for precise diagnostic methods in sheep. This study developed a loop-mediated isothermal amplification (LAMP) method targeting COX-1 and 28S rRNA genes to detect Sarcocystis tenella and Sarcocystis gigantea, respectively. The LAMP method exhibited high specificity, selectively amplifying target DNA sequences without cross-reactivity with closely related protozoa, such as Toxoplasma gondii and Neospora caninum. Detection limits were determined as 3 × 105 copies/L for S. tenella and 6 × 104 copies/L for S. gigantea, enabling sensitive identification of low-level infections. Comparative analysis with conventional PCR on sheep cardiac tissues demonstrated a higher LAMP detection rate (80.0% vs 66.7%). In conclusion, the LAMP method offers superior sensitivity to conventional PCR, allows visual confirmation of results, and provides a rapid diagnostic tool for identifying S. tenella and S. gigantea infection in sheep. However, due to the limitation of sample availability, we were unable to assess all Sarcocystis species that use sheep as intermediate hosts, which warrants further research.

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开发一种高度特异性的 LAMP 检测方法,用于检测绵羊中的天疱疮沙雷氏菌和巨型沙雷氏菌
绵羊沙眼衣原体感染给畜牧业造成了巨大的经济损失,而沙眼衣原体物种之间的遗传相似性突出表明了对绵羊精确诊断方法的需求。本研究开发了一种环介导等温扩增(LAMP)方法,以 COX-1 和 28S rRNA 基因为靶标,分别检测绵羊沙雷氏囊尾蚴(Sarcocystis tenella)和巨型沙雷氏囊尾蚴(Sarcocystis gigantea)。LAMP 方法具有高度特异性,可选择性地扩增目标 DNA 序列,不会与弓形虫和犬新孢子虫等密切相关的原生动物产生交叉反应。天牛的检测限被确定为 3 × 105 拷贝/升,巨细胞天牛的检测限被确定为 6 × 104 拷贝/升,从而能够灵敏地识别低水平感染。对绵羊心脏组织与传统 PCR 的比较分析表明,LAMP 的检出率更高(80.0% 对 66.7%)。总之,与传统 PCR 相比,LAMP 方法具有更高的灵敏度,可对结果进行肉眼确认,是鉴别绵羊天牛和吉丁虫感染的快速诊断工具。然而,由于样本供应的限制,我们无法评估所有以绵羊为中间宿主的沙眼衣原体种类,这值得进一步研究。
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来源期刊
Parasitology Research
Parasitology Research 医学-寄生虫学
CiteScore
4.10
自引率
5.00%
发文量
346
审稿时长
6 months
期刊介绍: The journal Parasitology Research covers the latest developments in parasitology across a variety of disciplines, including biology, medicine and veterinary medicine. Among many topics discussed are chemotherapy and control of parasitic disease, and the relationship of host and parasite. Other coverage includes: Protozoology, Helminthology, Entomology; Morphology (incl. Pathomorphology, Ultrastructure); Biochemistry, Physiology including Pathophysiology; Parasite-Host-Relationships including Immunology and Host Specificity; life history, ecology and epidemiology; and Diagnosis, Chemotherapy and Control of Parasitic Diseases.
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